Papaya (Carica papaya L.) is a soft-wooded herbaceous dicotyledonous plant that belongs to the family Caricacea. The family Caricaceae, contains thirty five latex containing species spread over six genera i.e. Carica (1 species), Cylicomorpha (2 species), Jarilla (3 species), Jacaratia (7 species), Horovitzia (1 species) and Vasconcellea (21 species) (Fuentes and Santamaria, 2014). Only the genera Carica; is used as a fruit tree and, now-a-days, also as a nutraceutical and medicinal plant (O'Hare and Williams, 2014). Papaya originated from Southern Mexico and Central America (Fuentes and Santamaria, 2014) and spread throughout the world. Papaya has nine pairs of chromosomes (2n=18) and approximately 372 MB genome size (Ming et al., 2008). Papaya ranks first among fruits consumed (Hui et al., 2001), and also ranks first in nutritional profile (Bari et al, 2006; Ming et al., 2008; Manshardt, 1992). The main five countries that have been producing papaya for the last 50 years are India, Nigeria, Brazil, Mexico and Indonesia (Kumar et al 2014).
The papaya ringspot virus (“PRSV”) is a member of the potyvirus group of plant viruses, which are pathogenic to several crop plants and is the most destructive disease in papaya. The name of the virus is taken from the ringed spots that form on the fruits of infected trees. Trees infected with PRSV develop symptoms such as mosaic and chlorosis of leaf lamina, water-soaked oily streaks on the petiole and upper part of the trunk, severe distortion of young leaves and formation of ring shaped spots on the fruits. If infected at seedling stage or within two months after planting, the tree might not bear any fruit leading to 100% losses. There are no wild species of papaya that show resistance to PRSV that can be used in breeding. Therefore, it is becoming more essential for plant breeders to develop plants that are resistant to infection from viruses, for example those from the Potyviridae family.
PRSV is transmitted by numerous species of aphids in a non-persistent manner to a limited host range of cucurbits and papaya. Currently the known solution for limiting loss to PRSV is by chemical control of vectors which is costly and does not provide 100% protection. The other solution is inducing resistance via transgenic route which is not only expensive but also not acceptable to consumers. Various attempts have been made to control or prevent infection of crops by PRSV, but these have met with limited success. U.S. Pat. No. 7,078,586 B2 (Gonsalves et. al 2006) describes a transgenic method of inducing resistance to the virus. This method involves the expression of nucleic acid sequences encoding coat protein of the mild mutant PRSV strain in transgenic papaya plants ‘Sunup’ which helps protect against infection of the PRSV to Hawaiin strains. However, the transgenic plants do not show resistance to PRSV strains outside Hawaii, such as Thailand and Jamaica PRSV strains (Gonsalves 2014). The variety ‘Sunup’ is a transgenic papaya ringspot virus resistant fruit crop which is currently grown in Hawaii, USA. In 2008, Ming et. al. first sequenced the papaya genome of the variety ‘Sunup’, having 3× genome coverage (Ming et al., 2008, Rachel, 2008). The papaya genome is reported to have about 13311 candidate genes (Ming et al., 2008) because of lack of gene duplications, which is quite different from other plant species. The genome is about three times the size of model plant Arabidopsis thaliana in terms of genome size, and contains far less number of disease resistance genes (Ming et al., 2008). It is possible that the genes in papaya might be involved in more than one function, as the estimated number of genes is quite low, compared to other species. Moreover, the papaya genome is highly euchromatic (Ming et al., 2008). Siar et al (2011), describe the production of PRSV resistant plants by backcrossing (BC) papaya plants following intergeneric hybridization between C. papaya and a Vasconcellea quercifolia with great difficulties. One PRSV-P resistant plant was produced after 114,839 seeds were dissected from 940 fruits of a back cross1 generation BC1). The above breeding methods have their own drawbacks, such as constant efforts to produce large number of plants after crossing for screening and efficient embryo culture requirements which are not easy to undertake. Moreover at present most markets are reluctant to accept and consume genetically modified fruits. Furthermore, the cost of de-regulating these transgenic papaya seeds can be very high. At present, there are no natural PRSV resistant Carica papaya species in the world that can be crossed easily (Gonsalves, 2014). Since, the genome of papaya has been already sequenced, specific gene related information is available by performing intensive data mining and comparative analysis with other sequenced crops. The inventors have developed novel methods to produce non-transgenic PRSV resistant papaya plants by inducing mutations in the papaya genome and screening of desirable mutants using Allele Detection Platform (ADP) a variant of the TILLING (Targeting Induced Local Lesions IN Genomes) method (Kumar. et al, 2014)
TILLING offers an alternative way to manipulate endogenous genes for the improvement of crops without transgenic method. TILLING is a reverse genetics technique that uses traditional chemical mutagenesis to create libraries of mutagenized individuals that are later subjected to sensitive molecular screenings to discover induced mutations in genes whose sequence is known. By means of TILLING new allelic variants of the gene of interest can be identified hence new genotypes with potentially high agronomic value can be isolated and directly transferred for commercialization after backcrossing to the parent line. Using this technique potyvirus resistant lines in various crops have been developed in crops such as in melons, tomato etc., as described by Robaglia and Caranta (2006), Neito et al (2006) Neito et al (2007), Ruffel et al (2002), Nicaise et al (2003) Gao et al, (2004) Kang et al (2005), Ruffel et al (2005) Piron et al (2010). For example, Piron et al (2010) teaches the use of TILLING to induce potyvirus resistance to Potato Virus Y (PVY) and Pepper Mottle Virus (PepMoV) in tomatoes. Very recently, Pyott et al (2016), utilized CRISPR-Cas9 technology to introduce sequence-specific deleterious point mutations at the eIF(iso)4e locus in Arabidopsis thaliana to successfully engineer complete resistance to Turnip mosaic virus (TuMV), a major pathogen in field-grown vegetable crops. However, up until now, these methods have not been used to induce mutations in papaya plants to develop PRSV resistant plants.
Therefore, embodiments of the present invention may ameliorate one or more of the above-mentioned problems:
Embodiments of the present invention may provide a papaya plant having increased resistance as compared to wild type papaya plants to the papaya ringspot virus due to a mutation in the eIF4e and/or eIF(iso)4e gene.
Another, embodiment of the present invention may provide a papaya plant having increased resistance as compared to the wild type papaya plant to the papaya ringspot virus due to a mutation in the eIF4e and/or eIF(iso)4e gene leading to non-functional eIF4e and/or eIF(iso)4E protein.
Another embodiment of the present invention may provide food and food products incorporating papaya fruits derived from a papaya plant having an increased resistance to the papaya ringspot virus caused by a mutation in the eIF4e and/or eIF(iso)4e gene and non-functional eIF4e and/or eIF(iso)4e proteins.
Yet another embodiment of the present invention may provide, a papaya plant having increased resistance to papaya ringspot virus created by the steps of obtaining plant material from a parent papaya plant, inducing at least one mutation in at least one copy of a eIF4e and/or eIF(iso)4e gene of the plant material by treating the plant material with a mutagen to create mutagenized plant material, culturing and growing the mutagenized plant material to produce progeny papaya plants, isolating DNA or RNA from progeny papaya plants and analyzing progeny papaya plants to detect at least one mutation in at least one copy of a eIF4e and/or eIF(iso)4e gene, leading to loss of function of the eIF4e and/or eIF(iso)4e protein and selecting progeny papaya plants and phenotyping plants that possess increased resistance to papaya ringspot virus; and repeating the cycle of culturing the progeny papaya plants to produce additional plants with increased resistance to papaya ringspot virus when compared to wild type papaya plants.
Some or all these and other objects of the invention can be achieved by way of the invention described here-in after.